Our general goal is to obtain detailed information on a molecular level about how proteins and lipids interact. We are undertaking a broad approach, utilizing a variety of biochemical and biophysical techniques, applied to several systems. Most of the proposed work involves an intensive study of rigorously purified components which have measurable biological activity. Several projects are proposed, all representing a continuation of work begun under the initial granting period. We are asking questions at several different levels concerning both the structural and functional aspects of protein-lipid interactions. Pyruvate oxidase and C55-isoprenoid alcohol phosphokinase are both lipid-requiring enzymes representative of the two operational classes of membrane enzymes, peripheral and intrinsic. Most of our efforts will be directed at pyruvate oxidase, a water soluble enzyme whic has several strong lipid-binding sites which are functionally significant. We hope to map the locations of these sites in relation to other ligand binding sites, obtain the peptide(s) involved, and determine in detail the mechanism of lipid activation. We have demonstrated that bilayer fluidity in vitro can be a critical parameter in determining the enzymatic rate of the kinase, an intrinsic membrane enzyme. Work is planned to quantitate the possible importance of such parameters of lipid hydration in relation to membrane enzyme activity. Bacitracin is a small peptide antibiotic whose structure is known and which forms a strong complex with polyisoprenyl pyrophophates. The complex formed with farnesyl pyrophosphate is not only biochemically relevant but will serve as an excellent model for protein-lipid studies centering around magnetic resonance techniques, including 13C NMR. In addition, the possibility of a "conformational coupling" between membrane lipids and proteins in intact E. coli is being studied in the context of the role of the membrane in energy transduction processes. Fluorescent probes and EPR spin probes, in addition to covalent labelling reagents, will be used to explore this problem.